There are two related projects in this Project. Project 1. Single cell transcriptional profiling of primitive mouse hematopoietic cell populations. We have determined that lineage profiles are determined for megakaryocytic, lymphocytes, neutrophils and monocytes are specified in the earliest populations of hematopoietic progenitor cells while the erythroid lineage emerges at the Common Myeloid Progenitor stage. We are have developed methods to prospectively isolate these populations for more in-depth study. Project 2. Defining the transcriptional and epigenetic profiles of differentiating mouse hematopoietic cells. We will determine the chromatin accessibility as well as the methylation and acetylation of histone proteins across the genome and correlated these with mRNA expression and DNA methylation. In mammals, the number of HSC per animal is remarkably similar between species despite great differences in the number of cells produced. Hematopoiesis has traditionally been modeled as a hierarchy in which the progeny of HSC become progressively committed to a branch (myeloid or lymphoid) and ultimately to a single lineage in a stepwise fashion. Recent single cell analyses and improved colony assays have shown that hematopoiesis follows a more fluid process where lineages are specified (at least at the transcriptional level) earlier in hematopoiesis than the original hierarchical model predicted. For example, we found that the human MEP population contains three subpopulations of lineage primed cells that could be prospectively separated by surface marker expression. In Project 1 we have performed single cell transcriptional profiling of primitive mouse hematopoietic cell populations and determine the stage of hematopoiesis where the transcriptional profiles of the erythroid and other lineages become specified. We have performed indexed single cell analyses to link these profiles for prospective sorting. Sorted cells are analyzed for their ability to differentiate into cells of one or more lineages and for identification of the epigenetic marks associated with mature hematopoietic. We have completed a single cell profile of over 10,000 cells each from primitive hematopoietic stem and progenitor cells (LSK), Common Myeloid Progenitors (CMP), Megakaryocyte/Erythroid progenitors (MEP) and Granulocyte/Monocyte progenitors (GMP). In these populations we have identified 25 distinct transcriptional profiles. We have shown that the erythroid lineage is the last to emerge, following the lymphoid, granulocytic and monocyte and lymphoid lineages. We are currently evaluating the epigenetics of these primitive erythroid progenitors. Project 2 is to define the transcriptional and epigenetic profiles of differentiating mouse hematopoietic cells and to test whether these patterns are established in more primitive cells. This ambitious, hypothesis generating goal is well beyond the capacities of any single lab and we have joined forces with the ValIdated Systematic IntegratiON of hematopoietic epigenomes consortium (VISION) which is a group of 10 different laboratories with the necessary wide range of expertise. Our involvement with VISION began in 2010 when we defined epigenetic changes associated with erythropoiesis in primary mouse hematopoietic cells. The rationale behind our choice of the mouse system was carefully considered. Our studies are intended to complement work being performed by ENCODE to define the epigenetic landscape in human hematopoietic cells. Mouse and human hematopoiesis, while highly conserved in some respects, also differ in many significant ways. By comparing the mouse and human epigenetic profiles, we will identify overlapping (common) patterns as well as distinct patterns that can be associated with the different properties of mouse and human erythropoiesis, which will generate more informed hypotheses than would be possible by studying hematopoiesis in a single species. For example, haploinsufficiency of ribosomal proteins in humans leads to a block in erythropoiesis resulting in Diamond Blackfan anemia, while haploinsufficiency of ribosomal proteins in mice is benign. Our group (Drs. Jens Lichtenberg and Elisabeth Heuston) have developed transcriptional, DNA methylation and histone modification profiles that complement data generated in the laboratories of Mitchell Weiss (St. Jude Childrens Research Hospital), Gerd Blobel (University of Pennsylvania), Jim Hughes (Oxford University) and Doug Higgs (Oxford University) to define the chromatin landscape during hematopoietic differentiation. These data will be integrated into a comprehensive 3D profile by the computational arm of VISION, which includes Jens Lichtenberg from our group, Berthold Gottgens (University of Cambridge), James Taylor (Johns Hopkins), Feng Yue (Penn State Hershey), Yu Zhang (Penn State) and Ross Hardison (Penn State). We have shown that the epigenetic profile of megakaryocytic is established in the most primitive hematopoietic stem and progenitor cells and is maintained during differentiation. In contrast, the epigenetic profile of erythroblasts is acquired during differentiation. We have found that the chromatin state profile varies from lineage to lineage and locus to locus. We hypothesize that each lineage has a specific epigenetic master regulator that is responsible for instituting the lineage specific program. We believe that identifying the differences (for example) in mouse and human RP gene regulation may identify a mouse like pathway that could be targeted to treat DBA patients.